Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Leukemia in a Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2961-2961
Author(s):  
Sarah M Greenblatt ◽  
Li Li ◽  
Christopher Slape ◽  
David Huso ◽  
Peter D. Aplan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Double Mutant ◽  
Tandem Duplication ◽  
Mutant Mice ◽  
Leukemic Cells ◽  
Internal Tandem Duplication ◽  
Cell Counts

Abstract Abstract 2961 Poster Board II-937 Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML), and provides a proliferative and survival advantage to leukemic cells. We have previously generated a knock-in mouse in which an 18-bp ITD mutation, isolated from a patient with AML, was inserted into the juxtamembrane domain of the murine FLT3 gene. Heterozygous FLT3WT/ITD mice develop a myeloproliferative disease, which progresses to mortality within 6 to 20 months. However, no sign of acute leukemia is observed over the lifetime of these mice, indicating that additional cooperating genetic events are required for leukemic progression. FLT3 activating mutations have been seen in MDS in about 5% of cases and an additional 5% of patients with MDS acquire FLT3 mutations as they progress to AML. One model of MDS has been developed by transgenic expression of the NUP98-HOXD13 (NHD13) fusion under the vav promoter. Mice expressing the NUP98-HOXD13 (NHD13) transgene develop a highly penetrant myelodysplastic syndrome (MDS) with about 50% eventually progressing to acute leukemia by 14 months. We wanted to generate a mouse model to see if FLT3/ITD mutations would cooperate with the NHD13 fusion to progress to overt leukemia. Double mutant mice (FLT3/ITD/NHD13) were generated by crossing heterozygous FLT3WT/ITD mice with mice expressing the NHD13 transgene. Strikingly, FLT3/ITD/NHD13 offspring (n=40) developed an acute leukemia with 100% penetrance and a median survival of 97 days. In contrast, NHD13 (n=20) and FLT3WT/ITD (n=20) littermates had median survivals of 385 and 410 days, respectively (Figure 1). Differential cell counts at the time of sacrifice showed elevated peripheral white blood cell counts in the double mutant mice (161.0±50.6 k/μl) compared to three-month-old wildtype (12.5 ±5.0 k/μl), NHD13(3.2 ±1.0 k/μl), or FLT3WT/ITD (7.9 ±3.5 k/μl) littermates. Organ sectioning and histological staining of double mutant mice showed leukemic infiltration of the spleen, liver, and brain. FLT3/ITD/NHD13 offspring developed a heterogeneous group of acute leukemias, with 46% developing ALL, 39% biphenotypic leukemia, and 15% AML as diagnosed by flow cytometry of the bone marrow and spleen. Transplantation experiments showed that the leukemia was able to engraft in lethally irradiated recipients, with disease occuring within 30 days post-transplantation. The disease was transferrable with as few as 1000 cells, and this ability was restricted to the B220+Mac-1−Gr-1- population. In order to identify the changes in gene expression responsible for leukemic transformation, RNA was isolated from the total bone marrow of young FLT3/ITD/NHD13, wildtype, NHD13, and FLT3WT/ITD littermates prior to the development of discernable disease and probed for over 43,000 coding and non-coding mouse sequences by Agilent 44K array. The rapid onset of acute leukemia in this model indicates the collaboration between hox gene dysregulation by a chromosomal translocation and altered signaling through the FLT3 receptor. In addition, since resistance to FLT3 inhibitors alone remains an important clinical issue, gene expression profiling of leukemic cells may help identify new molecular targets in collaborative signaling pathways. Disclosures: No relevant conflicts of interest to declare.

Modification of Gene Expression in Mesenchymal Stromal Cells of the Leukemia Patients during Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5065-5065
Author(s):  
Tamara Sorokina ◽  
Irina Shipounova ◽  
Alexey Bigildeev ◽  
Nina I. Drize ◽  
Larisa A. Kuzmina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Leukemic Cell ◽  
Expression Level ◽  
Leukemic Cells ◽  
Disease Manifestation ◽  
Relative Expression Level

Abstract Background In patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients. Aims To investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy. Methods 54 newly diagnosed cases (33 AML, 21 ALL) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used. Results At the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions. The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission. The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed. Conclusion Therefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Surveying Mutation of FLT3 Genes in Children with Acute Leukemia

2017 ◽  
Vol 2 (1) ◽  
pp. 7
Author(s):  
Maryam Sheikhi ◽  
Farhad Zaker ◽  
Gholamreza Javadi ◽  
Mehrdad Hashemi ◽  
Farnaz Razmkhah ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Point Mutation ◽  
Tandem Duplication ◽  
Leukemic Cells ◽  
Lymphoid Leukemia ◽  
Internal Tandem Duplication ◽  
Flt3 Mutation ◽  
Molecular Tests ◽  
Sequencing Method ◽  
Pcr Products

Background and purpose: Mutation of FMS like tyrosine kinase (flt3) gene causes uncontrolled proliferation of leukemic cells and a bad prognosis. The present study is aimed at implementing molecular tests to diagnose and screen the mutations in acute leukemia patients. Methodology: Totally, 91 children with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) were examined as to flt3 mutation, internal tandem duplication (ITD) mutation, and point mutation in exon 17 (e17). ITD mutation in flt3 receptor was carried out in exon 12&12 and intron 11. As to point mutation in e17, PCR products of the subjects after PCR on genomic DNA of them were examined using restriction enzyme (ECORV) and restriction fragment length polymorphism (RFLP). As to ITD positive, sequencing method was used. Findings: ITD mutation was observed in seven (7.7%) of acute leukemia patients and two (2.2%) patients were diagnosed with point mutation D835. Distribution in different subgroups of FAB was not identical. Conclusion: FLT3 mutation was highly prevalent in children with acute leukemia. Therefore, molecular diagnosis of these mutation, regardless of FAB categorization and before initiation of intervention, can be used to make better decision about therapeutic protocol.

scholarly journals Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3849-3858 ◽  
Author(s):  
Li Li ◽  
Obdulio Piloto ◽  
Ho Bao Nguyen ◽  
Kathleen Greenberg ◽  
Kogo Takamiya ◽  
...  
Keyword(s):  
Mouse Model ◽  
Acute Leukemia ◽  
Tandem Duplication ◽  
Myeloproliferative Disease ◽  
Internal Tandem Duplication ◽  
Hematopoietic Stem ◽  
Molecular Alterations ◽  
B Lymphoid

Abstract Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)–spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.

Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 865-865
Author(s):  
Sarah M Greenblatt ◽  
Li Li ◽  
Christopher Slape ◽  
Rachel L Novak ◽  
Amy S. Duffield ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Genetic Alterations ◽  
Molecular Pathways ◽  
Internal Tandem Duplication ◽  
Flt3 Mutations ◽  
Acute Myeloid

Abstract Abstract 865 Acute myeloid leukemia (AML) with myelodysplastic related changes represents 24–35% of all cases of AML and has a poor response to chemotherapy and a dismal prognosis. Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) receptor have been seen in 5% of myelodysplastic syndrome (MDS) cases, and an additional 10% of patients with MDS acquire FLT3 mutations during progression to AML. We have previously generated a knock-in mouse model in which an internal tandem duplication (ITD) mutation was inserted into the murine Flt3 gene, which induces a lethal myeloproliferative neoplasm, but not progression to overt leukemia. One mouse model of MDS involves the transgenic expression of a Nup98-HoxD13 (NHD13) fusion under the hematopoietic specific vav promoter. We bred the FLT3/ITD knock-in mice to the NHD13 transgenic mice to see if the two genetic alterations would cooperate. Strikingly, the FLT3/ITD-NHD13 mice on the FVB/N background developed acute leukemia with 100% penetrance and a mean survival of 95 ± 32 days (p<0.0001, n=20). This compares with a mean survival of 281 ± 94 days and 372 ± 84 days for the NHD13 alone and FLT3/ITD alone mice, respectively (both p<0.0001, n=20). FLT3/ITD-NHD13 mice generated on the C57Bl/6N background developed leukemia with a longer latency of 143 ± 37 days, but they still had a significantly shorter survival compared to the single mutants alone. Competitive repopulation experiments showed that leukemic bone marrow was able to engraft in lethally irradiated recipients, with 1:200 cells in the bulk bone marrow having the potential to generate leukemia in recipient mice. To determine the identity of the leukemic initiating cell (LIC), the bulk bone marrow was further sorted into the multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte/macrophage progenitor (GMP), and megakaryocytic/erythroid progenitor (MEP) populations and limiting dilution transplantation was performed for each group. The highest engraftment potential was found in the MPP population (1:100 cells) with much rarer LICs from the CMP and GMP. The MEP population did not engraft in any recipient mice even at the highest cell dose used of 100,000 sorted cells. RNA was extracted from the total bone marrow of mice 3 months prior to the development of disease and showed overexpression of HoxA7, HoxA9, HoxB4, HoxB6, HoxC4, and HoxC6 in the FLT3/ITD-NHD13 mice compared to the age-matched wildtype or FLT3/ITD mice. The overexpression of these genes appeared to be primarily driven by the NHD13 transgene, although there was also an increase in HoxA7 and HoxA9 expression observed in the FLT3/ITD mice. Since Hox genes are known to play an important role in stem cell self-renewal, we isolated RNA from 2-month-old littermates and stained for the cell surface markers characterizing the KSL, MPP, ST-HSC, and LT-HSC populations. FLT3/ITD-NHD13 and FLT3/ITD mice had a significant increase in the percentage of KSLs, long term HSCs, and short term HSCs. FLT3/ITD mice showed increased numbers of MPPs with a smaller but significant increase in the FLT3/ITD-NHD13 cohort. A significant number of AML patients with FLT3/ITD mutations present with loss of the wildtype allele of FLT3 and additional patients acquire this loss at the time of relapse. Hemizygosity at the FLT3 locus in FLT3/ITD mutant AML patients is associated with an even more adverse prognosis compared to patients with an intact wild-type allele. We observed spontaneous loss of heterozygosity (LOH) occurring in 100% of the FLT3/ITD-NHD13 mice and resulting in loss of the wildtype Flt3 allele. The frequent LOH seen in the FLT3/ITD-NHD13 mice provides additional evidence that this transgenic mouse serves as an accurate model to further explore the molecular pathways that underlie the development of leukemias with activating FLT3 mutations. This mouse model is also one of the first models of MDS-related AML and should provide a means to explore the molecular pathways that underlie these devastating hematopoietic neoplasms. Disclosures: Borowitz: BD Biosciences: Research Funding.

scholarly journals Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1951-1956
Author(s):  
ED Ball ◽  
J McDermott ◽  
JD Griffin ◽  
FR Davey ◽  
R Davis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Acute Leukemia ◽  
Cell Sorting ◽  
Lymphoblastic Leukemia ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Normal Bone

Monoclonal antibodies (MoAbs) have been prepared recently that recognize the three cell-surface receptors for the Fc portion of immunoglobulin (Ig), termed Fc gamma RI (MoAb 32.2), Fc gamma R II (MoAb IV-3), and Fc gamma R III (MoAb 3G8) that are expressed on selected subsets of non-T lymphocyte peripheral blood leukocytes. In the blood, Fc gamma R I is expressed exclusively on monocytes and macrophages, Fc gamma R II on granulocytes, mononuclear phagocytes, platelets, and B cells, and Fc gamma R III on granulocytes and natural killer (NK) cells. We have examined the expression of these molecules on normal bone marrow (BM) cells and on leukemia cells from the blood and/or BM in order to determine their normal ontogeny as well as their distribution on leukemic cells. BM was obtained from six normal volunteers and from 170 patients with newly diagnosed acute leukemia. Normal BM cells were found to express Fc gamma R I, II, and III with the following percentages: 40%, 58%, and 56%, respectively. Cell sorting revealed that both Fc gamma R I and Fc gamma R II were detectable on all subclasses of myeloid precursors as early as myeloblasts. Cell sorting experiments revealed that 66% of the granulocyte-monocyte colony-forming cells (CFU-GM) and 50% of erythroid burst-forming units (BFU-E) were Fc gamma R II positive with only 20% and 28%, respectively, of CFU-GM and BFU-E were Fc gamma R I positive. Acute myeloid leukemia (AML) cells expressed the three receptors with the following frequency (n = 146): Fc gamma R I, 58%; Fc gamma R II, 67%; and Fc gamma R III, 26% of patients. Despite the fact that Fc gamma R I is only expressed on monocytes among blood cells, AML cells without monocytoid differentiation (French-American-British [FAB]M1, M2, M3, M6) were sometimes positive for this receptor. However, Fc gamma R I was highly correlated with FAB M4 and M5 morphology (P less than .001). Fc gamma R II was also correlated with FAB M4 and M5 morphology (P = .003). Cells from 11 patients with acute lymphoblastic leukemia were negative for Fc gamma R I, but six cases were positive for Fc gamma R II and III (not the same patients). These studies demonstrate that Ig Fc gamma R are acquired during normal differentiation in the BM at or before the level of colony-forming units. In addition, we show that acute leukemia cells commonly express Fc gamma R.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1951-1956 ◽  
Author(s):  
ED Ball ◽  
J McDermott ◽  
JD Griffin ◽  
FR Davey ◽  
R Davis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Acute Leukemia ◽  
Cell Sorting ◽  
Lymphoblastic Leukemia ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Normal Bone

Abstract Monoclonal antibodies (MoAbs) have been prepared recently that recognize the three cell-surface receptors for the Fc portion of immunoglobulin (Ig), termed Fc gamma RI (MoAb 32.2), Fc gamma R II (MoAb IV-3), and Fc gamma R III (MoAb 3G8) that are expressed on selected subsets of non-T lymphocyte peripheral blood leukocytes. In the blood, Fc gamma R I is expressed exclusively on monocytes and macrophages, Fc gamma R II on granulocytes, mononuclear phagocytes, platelets, and B cells, and Fc gamma R III on granulocytes and natural killer (NK) cells. We have examined the expression of these molecules on normal bone marrow (BM) cells and on leukemia cells from the blood and/or BM in order to determine their normal ontogeny as well as their distribution on leukemic cells. BM was obtained from six normal volunteers and from 170 patients with newly diagnosed acute leukemia. Normal BM cells were found to express Fc gamma R I, II, and III with the following percentages: 40%, 58%, and 56%, respectively. Cell sorting revealed that both Fc gamma R I and Fc gamma R II were detectable on all subclasses of myeloid precursors as early as myeloblasts. Cell sorting experiments revealed that 66% of the granulocyte-monocyte colony-forming cells (CFU-GM) and 50% of erythroid burst-forming units (BFU-E) were Fc gamma R II positive with only 20% and 28%, respectively, of CFU-GM and BFU-E were Fc gamma R I positive. Acute myeloid leukemia (AML) cells expressed the three receptors with the following frequency (n = 146): Fc gamma R I, 58%; Fc gamma R II, 67%; and Fc gamma R III, 26% of patients. Despite the fact that Fc gamma R I is only expressed on monocytes among blood cells, AML cells without monocytoid differentiation (French-American-British [FAB]M1, M2, M3, M6) were sometimes positive for this receptor. However, Fc gamma R I was highly correlated with FAB M4 and M5 morphology (P less than .001). Fc gamma R II was also correlated with FAB M4 and M5 morphology (P = .003). Cells from 11 patients with acute lymphoblastic leukemia were negative for Fc gamma R I, but six cases were positive for Fc gamma R II and III (not the same patients). These studies demonstrate that Ig Fc gamma R are acquired during normal differentiation in the BM at or before the level of colony-forming units. In addition, we show that acute leukemia cells commonly express Fc gamma R.

scholarly journals CD7+ and CD56+ Myeloid/Natural Killer Cell Precursor Acute Leukemia: A Distinct Hematolymphoid Disease Entity

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2417-2428 ◽  
Author(s):  
Ritsuro Suzuki ◽  
Kazuhito Yamamoto ◽  
Masao Seto ◽  
Yoshitoyo Kagami ◽  
Michinori Ogura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Acute Leukemia ◽  
Natural Killer ◽  
Nk Cell ◽  
Bone Marrow Involvement ◽  
Leukemic Cells ◽  
Allogeneic Bone ◽  
Disease Entity ◽  
Cell Precursor

Abstract The disease spectrum of natural killer (NK) cell leukemias and lymphomas has recently been expanding with the continuing evolution in diagnostic concepts. We describe here seven cases of acute leukemia of conceivable myeloid and NK cell precursor phenotype in six men and one woman varying from 19 to 59 years of age (median, 46 years). Striking extramedullary involvement was evident at initial presentation, with peripheral lymphadenopathy and/or mediastinal masses. Two lacked any leukemic cells in the bone marrow at diagnosis. Using cytochemical myeloperoxidase staining, less than 3% of the leukemic cells showed positive reactivity. However, expression of CD7, CD33, CD34, CD56, and frequently HLA-DR, but not other NK, T-cell, and B-cell markers was observed. Cytoplasmic CD3 was detected in three of the cases by flow cytometry and in six by Northern blotting, suggesting an origin from common progenitors between the NK cell and myeloid lineages. All but one presented germline configurations of the T-cell receptor β and γ chain genes and Ig heavy chain gene. With regard to morphology, the cells were generally L2-shaped, with variation in cell size, round to moderately irregular nuclei and prominent nucleoli, pale cytoplasm, and a lack of azurophilic granules. Histopathologic examination of biopsied specimens of extramedullary tumors showed a lymphoblast-like morphology, implying the differential diagnostic problem from lymphoblastic lymphomas, especially in cases lacking bone marrow involvement. Three patients were successfully treated with chemotherapy for acute myeloid leukemia (AML), whereas three other patients proved refractory to chemotherapeutic regimens for lymphoid malignancies, although two responded to subsequent AML chemotherapy. However, despite intensive chemotherapy, including allogeneic bone marrow transplantation, most persued fatal courses within 41 months. These data suggested that the CD7+ and CD56+ myeloid/NK cell precursor acute leukemia might constitute a distinct biologic and clinical disease entity. Its recognition appears to be particularly important for the clinicopathologic evaluation of CD56+ hematolymphoid malignancies and the development of therapeutic approaches to such disease.

Characteristics of De Novo Acute Leukemia Patients with Glycosylphosphatidylinositol (GPI) Protein-Negative Population of Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4462-4462
Author(s):  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Masatoshi Okamoto ◽  
Kazuhiko Ikeda ◽  
Akiko Nakamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Remission Induction ◽  
Color Flow ◽  
Flow Cytometric ◽  
Number Of Patients

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be an acquired stem cell disorder affecting all hematopoietic lineages, which lack GPI-anchored membrane proteins, such as CD59, because of abnormalities in the phosphatidylinositol glycan-class A (PIG-A) gene. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome, which are considered as pre-leukemic states. In this study, to know some characteristics of patients with de novo acute leukemia, we investigated expression of CD59 in leukemic cells from 25 patients (female: male=8: 17; mean age ± standard deviation, 57.8 ± 19.5 years) with de novo acute leukemia by single-color flow cytometric analysis. In addition, the PIG-A gene from CD59− leukemic cells sorted by FACS Vantage in 3 patients with acute leukemia was examined by sequence analysis. All the patients had no past history of PNH. Based on the French-American-British criteria, the diagnosis and subtypes of acute leukemia were determined. The number of patients with subtypes M1, M2, M3, M4, M5, and M7 was 1, 14, 2, 4, 2, and 2, respectively. Two of the patients were classified into acute myeloid leukemia with trilineage myelodysplasia from morphological findings in bone marrow. Chromosomal analyses presented abnormal karyotypes in 14 of 25 patients. Flow cytometric analyses showed that leukemic cells from 16 of 25 patients (64%) had negative populations of CD59 expression and the proportion of the populations was 63.3 ± 25.7%, suggesting the possibility that CD59− leukemic cells from patients with de novo acute leukemia might be derived from PNH clones. In fact, the PIG-A gene analyses showed that monoclonal or oligoclonal PIG-A mutations in coding region were found in leukemic cells from 3 patients with CD59− leukemic cells and all of the clones with the PIG-A mutations were minor. Then, various clinical parameters, including rate of complete remission for remission-induction chemotherapy, peripheral blood, bone marrow blood, and laboratory findings, and results of chromosomal analyses were statistically compared between 2 groups of patients with (n=16) and without (n=9) CD59− leukemic cells. The reticulocyte counts (10.5 ± 13.0 x 104/μl) and proportions of bone marrow erythroblasts (17.5 ± 13.9%) in patients with only CD59+ leukemic cells were significantly higher than those (2.5 ± 1.7 x 104/μl, p&lt;0.05; and 5.6 ± 6.2%, p&lt;0.01, respectively) in patients with CD59− leukemic cells. The proportions of bone marrow blasts (69.3 ± 21.1%) in patients with CD59− leukemic cells were significantly higher than those (45.5 ± 19.3%, p&lt;0.02) in patients with only CD59+ leukemic cells. In conclusion, our findings indicate that leukemic cells derived from PNH clones may be common in de novo acute leukemia patients, suggesting that bone marrow failure may have already occurred in localized bone marrow even in de novo acute leukemia.

AMD3100 Mobilizes Acute Promyelocytic Leukemia Cells from the Bone Marrow into the Peripheral Blood and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 246-246 ◽  
Author(s):  
Bruno Nervi ◽  
Matthew Holt ◽  
Michael P. Rettig ◽  
Gary Bridger ◽  
Timothy J. Ley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Acute Promyelocytic Leukemia ◽  
Reporter Gene ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells

Abstract CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.

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